Reagents and methods for blocking non-specific interactions with nucleic acids

ABSTRACT

Methods and reagents for blocking non-specific interactions with nucleic acids are disclosed. In particular, the invention relates to multi-valent blockers comprising multiple negatively charged polymers or materials attached to a common scaffold and their use in blocking non-specific interactions with nucleic acids.

TECHNICAL FIELD

The present invention pertains generally to reagents and methods forblocking non-specific interactions with nucleic acids. In particular,the invention relates to multivalent blockers comprising multiplenegatively charged polymers or materials attached to a common scaffoldand their use in blocking non-specific interactions with nucleic acids.

REFERENCE TO SEQUENCE LISTING

The material in the accompanying sequence listing is hereby expresslyincorporated by reference in its entirety into this application. Theaccompanying sequence listing text file, named ENBIO001WO_SEQLIST.TXT,was created on Jan. 22, 2019, and is 1 KB in size. The content of thesequence listing is hereby expressly incorporated by reference in itsentirety.

BACKGROUND

Since the discovery of the structure of DNA by Watson and Crick, medicaland biological fields have advanced significantly. Groundbreakinginnovations have leveraged the unique biological roles and chemicalproperties of nucleic acids. Chemically tailored nucleic acids (e.g.,DNA, siRNA, nucleic acid derivatives) permit the efficient transfer ofnew genetic information into the target cells as a powerful therapeuticstrategy [1]. Gene editing methods such as Crispr-Cas9 use specializedRNA-guided protein machinery to delete, insert, and directly modify DNAfor research and therapeutic applications. In addition, bespoke nucleicacid sequences like aptamers form unique conformational structures tobind target proteins with high specificity and affinity as a promisingalternative to traditional antibody therapeutics [2]. Thus, it isevident that nucleic acid-based technologies are foundational to manyemerging therapies, and prevention of non-specific binding to thesenucleic acid materials is critical for enhancement of drug efficacy andreduction of off-target effects.

On the other hand, nucleic acid technologies also serve as powerfulanalytical tools for biomedical research and diagnostics. Indeed,nucleic acids are widely-used as barcodes in multiplex analysis ofbiological specimens. For instance, Nanostring has successfully usedfluorescent DNA barcoding to develop molecular diagnostic assays such astheir nCounter panel [3]. Other companies such as Somalogic use nucleicacid aptamers as antibody surrogates to create impressively large (>1300members) protein arrays. These innovations successfully use nucleic acidbarcoding to greatly expand the multiplexibility of analytical assays[4]. Prevention of non-specific binding to these nucleic acid barcodesis thus essential for maintaining high specificity in such multiplexassays.

Apart from their use as barcodes, nucleic acids have been used tosubstantially enhance the sensitivity of analytical assays. Immuno-PCRleverages the exponential amplification power of polymerase to augmentsignal amplification to achieve close to single-molecule-levelsensitivity. Moreover, newer generations of PCR-based immunoassays havebeen devised, such as antibody detection by agglutination-PCR (ADAP), toimprove sensitivity while simplifying assay workflow [5]. Thisinnovation opens the possibility of detecting disease-relevantbiomarkers at an earlier stage, increasing treatment options andeffectiveness to improve patient outcomes. Notably, even very weaknon-specific binding of nucleic acid materials is likely to causedetectable background signals in these highly sensitive assays, therebycompromising assay specificity.

Additionally, many nucleic acid-based assays use the ability of nucleicacids to rapidly and faithfully bind to its reverse-complement as amechanism of detection. For instance, complementary nucleic acid strandscould be installed on solid surface, capturing its reverse complementmolecule present in a biological specimen through a hybridizationreaction. Then, a secondary reporter can be added to generate signals.These secondary reporters could generate signals through diverse means,such as fluorescent signals (by tagging fluorescent tags), electronicsignals (by tagging molecules capable of generating electrons) ormagnetic signals (by tagging magnetic particles). The assay workflowsmay or may not require washing steps to remove unbound secondaryreporter. Companies such as Affimetrix (fluorescent arrays), T2Biosystems (magnetic signals) [6], Genmark (electric signals) [7], DNAe(electric signals) employ such mechanisms in their products. Genomicelements can also be directly labelled in biospecimen to simultaneousquantify gene expression and localization. A widely-used version of thisapproach is fluorescence in situ hybridization (FISH), which is commonlyemployed for tissue slices. Finally, recent technology advanced bycompany such as InCellDx also allows fluorescence in situ hybridizationon intact cells, thereby allowing downstream flow cytometry analysis[8]. As a whole, these assays rely on highly-specific interactionsbetween nucleic acids and their reverse-complementary strands to achievedetection of biomolecules in a complex environment. As a result,prevention of nonspecific binding to nucleic acid materials areinstrumental to maintain these assays' specificity.

Finally, advancement of next-generation sequencing (NGS) methods hasrevolutionized biomedical research. A critical component of NGS islibrary preparation. Library preparation refers to a process from whichinitial nucleic acid materials in a biospecimen is converted to NGScompatible format. Today's technologies permit library preparation fromultra-low sample inputs (e.g. 10 pg RNA input), and even down to thesingle cell levels in some cases [9]. Prevention of non-specific bindingof nucleic acid materials would greatly increase the library preparationyield (e.g. increase the percentage of single cells from which asuccessful NGS library could be obtained by preventing loss of genomicmaterials due to non-specific binding to lab consumables). In addition,many NGS methods require binding of nucleic acid library materials ontoa solid support (e.g. 454 (Roche), SOLiD (Thermo Fisher), GeneReader(Qiagen), Ion Torrent (Thermo Fisher), Illumina, and Complete GenomicsBGI). The solid support could be a microarray array, a bead, a glassslide or a structured solid substrate. Again, non-specific anchoring ofgenomic materials onto these solid supports would create biased andunwanted background signals in the sequencing process, and should beprevented. Furthermore, recent advancement of analytical methods allowsuse of nucleic acid-barcoded antibodies to achieve simultaneoussingle-cell analysis of protein and RNA expression [9]. Similarly,nucleic acid-barcoded MHC tetramers have been used to interrogate T cellreceptor specificity and corresponding gene expression simultaneously[10]. These methods again largely require a method to reducenon-specific binding of nucleic acid barcoded materials onto cells beinganalyzed, thereby reducing unwanted background signals.

However, several universal challenges remain for prevention ofnon-specific nucleic acid binding. Nucleic acids bear a strong negativecharge due to phosphate groups that compose the molecule's backbone.This negative charge confers an electrostatic affinity to positivelycharged molecules in a sequence- and barcode-independent manner. Thischarge affinity is a major source of unwanted non-specific interactions,leading to (1) nucleic acid therapies or diagnostic probes sequesteredin undesired locations, thereby reducing actual dosing in the targetcells (2) nucleic acid analytical reagents creating nonspecific andoff-target signals, leading to false positives and negatives (3)reduction of yield in single cell or ultra-low sample inputnext-generation sequencing experiments. These issues greatly reduce thereliability of therapeutics, research or diagnostics tools based onnucleic acid materials. The current state of the art employs excessinert single-stranded or double-stranded nucleic acids as blockingagents to prevent non-specific interactions between precious functionalnucleic acids and other molecules [9]. Common sources of these state ofthe art blocking agents are extracted DNA or RNA from salmon sperm or E.coli. However, in many circumstances, these biologically derivedblocking agents suffer from varying compositions from batch to batch,and still cannot fully prevent non-specific binding in many cases [9].In addition, charged detergents such as sodium dodecyl sulfate (SDS) anddextran sulfate are also used to achieve a similar result [9].Nevertheless, it is widely reported that these detergents could eitherdenature protein components in the system, or could inhibit downstreamapplications (e.g. PCR), thereby introducing their own interferences. Insummary, though these important state of the art methods havefacilitated the use of nucleic acid technologies, their abilities toabrogate non-specific binding of nucleic acids materials are limited. Itis well-known in the field that the applications of nucleic acidsmaterials mentioned above still suffer from specificity and off-targetissues. Thus, there remains a need for better methods of blockingnon-specific interactions between nucleic acids and other molecules.

SUMMARY

The present invention is based on the development of reagents andmethods for blocking non-specific interactions with nucleic acids.

In one aspect, the invention includes method of blocking non-specificinteractions with a nucleic acid of interest in a sample, the methodcomprising contacting the sample with a multivalent blocker, saidmultivalent blocker comprising at least two negatively charged polymerslinked to a scaffold, wherein the multivalent blocker binds topositively charged compounds or materials in the sample, therebyblocking non-specific interactions with the nucleic acid of interest.

In certain embodiments, 2-10 or more negatively charged polymers may belinked to the scaffold, including any number of negatively chargedpolymers in this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenegatively charged polymers. The polymers linked to the scaffold, forexample, may comprise one or more negatively charged functional groupssuch as, but not limited to, carboxylate, sulfate, and phosphate groups.The negatively charged polymers can be linked to the scaffold covalentlyor noncovalently.

In certain embodiments, the polymers linked to the scaffold are nucleicacids. For example, 2-10 or more nucleic acids may be linked to thescaffold, including any number of nucleic acids in this range, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acids.

In other embodiments the negatively charged polymer is a poly(acrylicacid) polymer. For example, 2-10 or more negatively charged poly(acrylicacid) polymers may be linked to the scaffold, including any number ofnegatively charged poly(acrylic acid) polymers in this range, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more poly(acrylic acid) polymers.

In certain embodiments the scaffold is a dendrimer (e.g., polyamidoamine(PAMAM) dendrimer), a multi-armed polyethylene glycol (PEG), ananoparticle (e.g., gold nanoparticles), or streptavidin.

In certain embodiments, the scaffold is spherical (e.g., beads,pellets), nonspherical (e.g., non-spherical nano- and micro-scaleparticles), linear (e.g., linear polymers or fibers), branched (e.g. 2or more branches with binding sites for negatively charge polymers), orplanar (e.g., thin sheet, membrane, or plate). The scaffold may range insize from about 0.3 nm to about 5 nm in length, including any length inthis range such as 0.3 nm, 0.4 nm, 0.5 nm, 0.75 nm, 1 nm, 1.5 nm, 2 nm,2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, or 5 nm.

In certain embodiments, the nucleic acid of interest is a nucleic acidprobe comprising a detectable label. In another embodiment, themultivalent blocker further comprises the same detectable label as thenucleic acid probe. The detectable label can be, for example, afluorescent, bioluminescent, or chemiluminescent label, and may beattached to the scaffold or a negatively charged polymer linked to thescaffold.

In certain embodiments, the method further comprises performing realtime quantitative polymerase chain reaction (RT-PCR), microarrayanalysis, fluorescent in situ hybridization (FISH), a NanoString assay,next generation sequencing, fluorescence resonance energy transfer(FRET), T2 magnetic resonance (T2MR), antibody-detection byagglutination PCR (ADAP), CRISPR-CAS9 genome editing, or transfectionwhile blocking the non-specific interactions with the nucleic acid ofinterest.

In certain embodiments, the nucleic acid of interest is conjugated to anagent such as, but not limited to, an antibody, an antigen, a peptide, aprotein, a lipid, a carbohydrate, a small molecule, a nanoparticle, or acationic molecule. In certain embodiments, the nucleic acid of interestis DNA or RNA. For example, the nucleic acid of interest may be an RNAselected from the group consisting of messenger RNA (mRNA), transfer RNA(tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), microRNA(miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), and longnoncoding RNA (lncRNA).

In another example, the nucleic acid of interest is a DNA aptamer or RNAaptamer. In another aspect, the invention includes a compositioncomprising a multivalent blocker comprising at least two negativelycharged polymers or materials linked to a scaffold, as described herein.

In certain embodiments, the composition comprises a multivalent blockercomprising at least one nucleic acid linked to the scaffold, wherein atleast one nucleic acid comprises the nucleotide sequence of SEQ ID NO:1(5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ ID NO:1),or a sequence displaying at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%sequence identity thereto, wherein the multivalent blocker is capable ofblocking nonspecific interactions with a nucleic acid of interest in asample.

In another embodiment, the composition further comprises one or morenucleic acids of interest.

In another embodiment, the composition further comprises one or morereagents for performing real time quantitative PCR, microarray analysis,fluorescent in situ hybridization (FISH), a NanoString assay, nextgeneration sequencing, fluorescence resonance energy transfer (FRET), T2magnetic resonance (T2MR), antibody-detection by agglutination PCR(ADAP), or CRISPR-CAS9 genome editing, or transfection.

In another embodiment, the invention includes a kit comprising amultivalent blocker described herein and instructions for using themultivalent blocker for blocking non-specific interactions with anucleic acid if interest in a sample. In another embodiment, the kitfurther comprises one or more reagents for performing real timequantitative PCR, microarray analysis, fluorescent in situ hybridization(FISH), a NanoString assay, next generation sequencing, fluorescenceresonance energy transfer (FRET), T2 magnetic resonance (T2MR),antibody-detection by agglutination PCR (ADAP), or CRISPR-CAS9 genomeediting, or transfection.

Preferred alternatives of the invention include the following.

-   -   1. A method of blocking non-specific interactions with a nucleic        acid of interest in a sample, the method comprising contacting        the sample with a multivalent blocker, said multivalent blocker        comprising at least two negatively charged polymers linked to a        scaffold, wherein the multivalent blocker binds to positively        charged compounds or materials in the sample, thereby blocking        non-specific interactions with the nucleic acid of interest.    -   2. The method of alternative 1, wherein 2-10 negatively charged        polymers are linked to the scaffold.    -   3. The method of alternative 1 or 2, wherein 5 negatively        charged polymers are linked to the scaffold.    -   4. The method of anyone of alternatives 1-3, wherein the        negatively charged polymers linked to the scaffold are nucleic        acids, poly(acrylic acid) polymers, polysaccharides, or        peptides.    -   5. The method of alternative 4, wherein the nucleic acids linked        to the scaffold are RNA or DNA.    -   6. The method of any one of alternatives 1-5, wherein the        negatively charged polymers linked to the scaffold comprise        negatively charged functional groups.    -   7. The method of alternative 6, wherein the negatively charged        functional groups are selected from the group consisting of        carboxylate, sulfate, and phosphate.    -   8. The method of anyone of alternatives 1-7, wherein the        scaffold comprises a dendrimer, a protein, a multiarmed        polyethylene glycol (PEG), or a nanoparticle.    -   9. The method of alternative 8, wherein the dendrimer is a        polyamidoamine (PAMAM) dendrimer.    -   10. The method of alternative 8, wherein the protein is        streptavidin or avidin.    -   11. The method of alternative 8, wherein the nanoparticle is a        gold nanoparticle.    -   12. The method of anyone of alternatives 1-11, wherein the        scaffold is spherical, nonspherical, linear, branched, or        planar.    -   13. The method of anyone of alternatives 1-12, wherein the        scaffold has a size ranging from about 0.3 nm to about 5 nm in        length.    -   14. The method of anyone of alternatives 1-13, wherein the        negatively charged polymers are linked to the scaffold        covalently or noncovalently.    -   15. The method of anyone of alternatives 1-14, wherein the        nucleic acid of interest is a nucleic acid probe comprising a        detectable label.    -   16. The method of alternative 15, wherein the multivalent        blocker further comprises the same detectable label as the        nucleic acid probe.    -   17. The method of alternative 15 or 16, wherein the detectable        label is a fluorescent, bioluminescent, or chemiluminescent        label.    -   18. The method of anyone of alternatives 1-17, further        comprising performing real time quantitative polymerase chain        reaction (RT-PCR), microarray analysis, fluorescent in situ        hybridization (FISH), a NanoString assay, next generation        sequencing, fluorescence resonance energy transfer (FRET), T2        magnetic resonance (T2MR), antibody-detection by agglutination        PCR (ADAP), CRISPR-CAS9 genome editing, or transfection while        blocking the non-specific interactions with the nucleic acid of        interest with the multivalent blocker.    -   19. The method of anyone of alternatives 1-18, wherein the        nucleic acid of interest is conjugated to an agent.    -   20. The method of alternative 19, wherein the agent is an        antibody, an antigen, a peptide, a protein, a lipid, a        carbohydrate, a small molecule, a nanoparticle, or a cationic        molecule.    -   21. The method of anyone of alternatives 1-20, wherein the        nucleic acid of interest is DNA or RNA.    -   22. The method of alternative 21, wherein the RNA is selected        from the group consisting of messenger RNA (mRNA), transfer RNA        (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA),        microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA        (snRNA), and long noncoding RNA (lncRNA).    -   23. The method of alternative 21, wherein the nucleic acid of        interest is a DNA aptamer or RNA aptamer.    -   24. A composition comprising a multivalent blocker comprising at        least two negatively charged polymers or materials linked to a        scaffold.    -   25. The composition of alternative 24, wherein the polymers        linked to the scaffold are nucleic acids.    -   26. The composition of alternative 25, wherein at least one        nucleic acid is selected from the group consisting of: a) a        nucleic acid comprising a nucleotide sequence of        (5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ        ID NO:1); and b) a nucleic acid comprising a nucleotide sequence        having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,        93, 94, 95, 96, 97, 98, or 99% identity to the sequence of        (5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ        ID NO:1).    -   27. The composition of anyone of alternatives 24-26, wherein        2-10 nucleic acids are linked to the scaffold.    -   28. The composition of anyone of alternatives 24-27, wherein 5        nucleic acids are linked to the scaffold.    -   29. The composition of anyone of alternatives 24-28, further        comprising reagents for performing real time quantitative        polymerase chain reaction (RT-PCR), microarray analysis,        fluorescent in situ hybridization (FISH), a NanoString assay,        next generation sequencing, fluorescence resonance energy        transfer (FRET), T2 magnetic resonance (T2MR),        antibody-detection by agglutination PCR (ADAP), or CRISPR-CAS9        genome editing, or transfection.    -   30. A kit comprising the composition of anyone of alternatives        24-29 and instructions for using a multivalent blocker for        blocking non-specific interactions with nucleic acids in a        sample.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that a traditional nucleic acid blocker could not bindnon-specific binding substances with high enough avidity to eliminatenon-specific binding. The multi-valent negative charge blocker couldbind with much higher avidity, thereby efficiently shut downnon-specific binding interactions. The core of multivalent blocker couldbe any materials that are capable of holding 2 or more nucleic acidtogether.

FIG. 2 shows that the multivalent negative charge blocker could usenucleic acid (e.g. DNA, RNA) as the negative charge source.Alternatively, it could also use other negative charge polymer as anegative charge sources such as polyacrylic acid.

FIG. 3 shows that the multivalent negative charge blocker can be furtherdecorated with desired modification to better mimic the actual nucleicacid reagent in the biological system. For instance, one can decoratethe multivalent nucleic acid with biotin label, digoxigenin label,fluorophore label. In this way, the multi-valent blocker could alsoprevent non-specific binding onto the label.

FIG. 4 shows that the multivalent nucleic acid blocker can be incubatedwith nucleic acids in advance to pre-occupy non-specific binding sites.Alternatively, one can introduce the target nucleic acid (e.g. aptamer,reporter labelled nucleic acid, nucleic acid conjugates, etc.) alongsidethe multivalent nucleic acid blocker to prevent non-specific bindingfrom happening.

FIG. 5 shows that the multivalent nucleic acid blocker can besynthesized by conjugating a thiolated nucleic acid onto a dendrimerscaffold.

FIG. 6 shows tests for blocking the efficiency of various blockers. Thedata demonstrated multivalent blocker completely eliminated non-specificbackground signals when assaying patient serum containing interferingsubstances.

FIG. 7 shows tests of the impact of different blockers on testperformance. A target analyte was serially diluted and assayed by ADAP.The data showed that the multivalent blocker did not affect assayperformance, whereas dextran sulfate reduced signal intensities andassay sensitivities.

FIG. 8 shows tests of the conjugation strategy on blocking efficiency.The data demonstrated various core materials could be used for themultivalent blocker. The results unambiguously showed blockingefficiency depended on multivalence.

FIG. 9 shows tests of the dependence of blocking efficiency on valency.The data demonstrated bivalency alone could boost blocking efficiencysignificantly, and the effect plateaus at 5 DNA per blocker.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology and recombinant DNA techniques, within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Nucleic Acid Detection: Methods and Protocols (Methods in MolecularBiology, D. M. Kolpashchikov and Y. V. Gerasimova eds., Humana Press,2013); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir andC. C. Blackwell eds., Blackwell Scientific Publications); T. E.Creighton, Proteins: Structures and Molecular Properties (W.H. Freemanand Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,Inc., current addition); M. R. Green and J. Sambrook Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory Press, 4th edition,2012); Methods In Enzymology (S. Colowick and N. Kaplan eds., AcademicPress, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby expressly incorporated by reference in theirentireties.

1. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a nucleic acid” includes a mixture of two or nucleicacids, and the like.

The term “about,” particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used herein to include a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. It also includes modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing nonnucleotidic backbones,for example, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA. There is no intendeddistinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms will be used interchangeably. Thus, these terms include, forexample, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, DNA:RNAhybrids, and hybrids between PNAs and DNA or RNA, and also include knowntypes of modifications, for example, labels which are known in the art,methylation, “caps,” substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide or oligonucleotide.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

As used herein, the term “binding pair” refers to first and secondmolecules that specifically bind to each other, such as a ligand and areceptor, an antigen and an antibody, or biotin and streptavidin.“Specific binding” of the first member of the binding pair to the secondmember of the binding pair in a sample is evidenced by the binding ofthe first member to the second member, or vice versa, with greateraffinity and specificity than to other components in the sample. Thebinding between the members of the binding pair is typicallynoncovalent.

As used herein, a “solid support” refers to a solid surface such as amagnetic bead, non-magnetic bead, microtiter plate well, glass plate,nylon, agarose, acrylamide, and the like.

“Substantially purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, peptide composition)such that the substance comprises the majority percent of the sample inwhich it resides. Typically, in a sample, a substantially purifiedcomponent comprises 50%, preferably 80%-85%, more preferably 90-95% ofthe sample. Techniques for purifying polynucleotides and polypeptides ofinterest are well-known in the art and include, for example,ion-exchange chromatography, affinity chromatography and sedimentationaccording to density.

By “isolated” is meant, when referring to a protein, polypeptide orpeptide, that the indicated molecule is separate and discrete from thewhole organism with which the molecule is found in nature or is presentin the substantial absence of other biological macro molecules of thesame type. The term “isolated” with respect to a nucleic acid is anucleic acid molecule devoid, in whole or part, of sequences normallyassociated with it in nature; or a sequence, as it exists in nature, buthaving heterologous sequences in association therewith; or a moleculedisassociated from the chromosome.

As used herein, the term “probe” or “oligonucleotide probe” refers to apolynucleotide, as defined above, that contains a nucleic acid sequencecomplementary to a nucleic acid sequence present in the target nucleicacid analyte. The polynucleotide regions of probes may be composed ofDNA, and/or RNA, and/or synthetic nucleotide analogs. Probes may belabeled in order to detect the target sequence. Such a label may bepresent at the 5′ end, at the 3′ end, at both the 5′ and 3′ ends, and/orinternally. The “oligonucleotide probe” may contain at least onefluorescer and at least one quencher. Quenching of fluorophorefluorescence may be eliminated by exonuclease cleavage of thefluorophore from the oligonucleotide (e.g., TaqMan assay) or byhybridization of the oligonucleotide probe to the nucleic acid targetsequence (e.g., molecular beacons). Additionally, the oligonucleotideprobe will typically be derived from a sequence that lies between thesense and the antisense primers when used in a nucleic acidamplification assay.

The terms “hybridize” and “hybridization” refer to the formation ofcomplexes between nucleotide sequences which are sufficientlycomplementary to form complexes via Watson-Crick base pairing. Where aprimer “hybridizes” with target (template), such complexes (or hybrids)are sufficiently stable to serve the priming function required by, e.g.,the DNA polymerase to initiate DNA synthesis.

It will be appreciated that the hybridizing sequences need not haveperfect complementarity to provide stable hybrids. In many situations,stable hybrids will form where fewer than about 10% of the bases aremismatches, ignoring loops of four or more nucleotides. Accordingly, asused herein the term “complementary” refers to an oligonucleotide thatforms a stable duplex with its “complement” under assay conditions,generally where there is about 90% or greater homology.

The term “antibody” encompasses polyclonal and monoclonal antibodypreparations, as well as preparations including hybrid antibodies,altered antibodies, chimeric antibodies and, humanized antibodies, aswell as: hybrid (chimeric) antibody molecules (see, for example, Winteret al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)2and F(ab) fragments; Fv molecules (noncovalent heterodimers, see, forexample, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; andEhrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules(sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g.,Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al. (2012)Methods Mol Biol 911:15-26; dimeric and trimeric antibody fragmentconstructs; minibodies (see, e.g., Pack et al. (1992) Biochem31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanizedantibody molecules (see, e.g., Riechmann et al. (1988) Nature332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K.Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, anyfunctional fragments obtained from such molecules, wherein suchfragments retain specific-binding properties of the parent antibodymolecule.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring toan antigen or allergen, refers to a binding reaction that isdeterminative of the presence of the antigen or allergen in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular antigen at least two times the background and do notsubstantially bind in a significant amount to other antigens present inthe sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular antigen. For example, polyclonal antibodies raised to anantigen from specific species such as rat, mouse, or human can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with the antigen and not with otherproteins, except for polymorphic variants and alleles. This selectionmay be achieved by subtracting out antibodies that cross-react withmolecules from other species. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularantigen. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane. Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically, a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

As used herein, a “biological sample” refers to a sample of cells,tissue, or fluid isolated from a subject, including but not limited to,for example, blood, plasma, serum, fecal matter, urine, bone marrow,bile, spinal fluid, lymph fluid, samples of the skin, externalsecretions of the skin, respiratory, intestinal, and genitourinarytracts, tears, saliva, milk, cells (e.g., epithelial and endothelialcells, fibroblasts, and macrophages), muscles, joints, organs (e.g.,liver, lung, spleen, thymus, kidney, brain, or lymph node), biopsies andalso samples of in vitro cell culture constituents including but notlimited to conditioned media resulting from the growth of cells andtissues in culture medium, e.g., recombinant cells, and cell components.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,semiconductor nanoparticles, dyes, metal ions, metal sols, ligands(e.g., biotin, strepavidin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof which is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels which may be used in the practice of the invention include,but are not limited to, SYBR green, SYBR gold, a CAL Fluor dye such asCAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL FluorRed 610, and CAL Fluor Red 635, a Quasar dye such as Quasar 570, Quasar670, and Quasar 705, an Alexa Fluor such as Alexa Fluor 350, Alexa Fluor488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647,and Alexa Fluor 784, a cyanine dye such as Cy 3, Cy3.5, Cy5, Cy5.5, andCy7, fluorescein, 2′, 4′, 5′, 7′-tetrachloro-4-7-dichlorofluorescein(TET), carboxyfluorescein (FAM),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),hexachlorofluorescein (HEX), rhodamine, carboxy-Xrhodamine (ROX),tetramethyl rhodamine (TAMRA), FITC, dansyl, umbelliferone, dimethylacridinium ester (DMAE), Texas red, luminol, NADPH, horseradishperoxidase (HRP), and α-β-galactosidase.

2. Modes of Carrying Out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

The present invention is based on the discovery of reagents and methodsfor blocking non-specific interactions with nucleic acids. The inventorshave shown that multivalent blockers comprising at least two negativelycharged polymers linked to a scaffold are effective in blockingnon-specific interactions with nucleic acids in a sample (Example 1). Inparticular, the inventors designed multivalent blockers comprisingmultiple (2 or more) nucleic acids or negatively charged acrylic acidpolymers linked to scaffolds made up of dendrimers, streptavidin, orgold nanoparticles and showed that such multivalent blockers weresignificantly more effective in blocking non-specific interactions withnucleic acids than single-stranded or double-stranded nucleic acidscommonly used as blocking agents (Example 1).

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding such multivalent blockers andtheir use in various applications for reducing interference fromnon-specific interactions with nucleic acids.

A. Multivalent Blockers

Multivalent blockers for blocking non-specific interactions with nucleicacids comprise at least two negatively charged polymers linked to ascaffold. Such multivalent blockers bind to positively charged compoundsor materials in a sample, thereby blocking non-specific interactionswith nucleic acids of interest. The multivalent blockers of theinvention will find use with various nucleic acid-based techniques toreduce undesired nonspecific interactions with nucleic acids.

The scaffold in the multivalent blocker provides a structure upon whichthe negatively charged polymers can associate or attach. Scaffolds mayhave a variety of geometric shapes, including spherical (e.g., beads,pellets), nonspherical (e.g., non-spherical nano- and micro-scaleparticles), linear (e.g., linear polymers or fibers), branched (e.g. 2or more branches with binding sites for negatively charge polymers), orplanar (e.g., thin sheet, membrane, or plate). Exemplary scaffoldmaterials include dendrimers (e.g., PAMAM), multi-armed polyethyleneglycol (PEG), metal nanoparticles (e.g., nanoparticles comprisingbiocompatible metals such as gold, silver palladium, platinum, ortitanium, or metal alloys or oxides thereof), or proteins (e.g.,streptavidin). Additionally, a coating may be added to the surface of ascaffold to facilitate attachment of negatively charged polymers to thesurface.

In certain embodiments, the scaffold comprises a dendrimer. Dendrimersare symmetric, spherical, highly branched compounds made up of a seriesof branches extending from an inner core. The compounds are classifiedby generation, which refers to the number of branching synthesis cyclesthat are performed to produce them. Dendrimers of higher generation havemore cycles of branching and higher molecular weights. A variety ofdendrimers are suitable for use as scaffolds in multivalent blockers,including, but not limited to, polylysine, poly(amidoamine) (PAMAM),poly(propylene imine) (PPI or DAB), and poly(etherhydroxylamine) (PEHAM)dendrimers of various generations. For a description of dendrimers andmethods of synthesizing and conjugating them, see, e.g., Dendrimers andOther Dendritic Polymers (Wiley Series in Polymer Science, J. M. J.Fréchet and D. A. Tomalia eds., Wiley, 2002), Dendrimers (Topics inCurrent Chemistry, Fritz Vogtle ed., Springer, 1998), Dendrimers:Synthesis, Applications and Role in Nanotechnology (Chemical EngineeringMethods and Technology, H. B. Harris and B. L. Turner eds., Nova SciencePub Inc, 2013), Kalhapure et al. (2015) Pharm. Dev. Technol.20(1):22-40, Sato et al. (2013) Molecules. 18(7):8440-8460, Lallana etal. (2012) Pharm. Res. 29(4):902-921, Caminade et al. (2012) Molecules17(11):13605-21, Wang et al. (2012) Curr. Med. Chem. 19(29):5011-5028,Arseneault et al. (2015) Molecules 20(5):9263-9294, Walter et al. (2012)Chem. Soc. Rev. 41(13):4593-4609, and Najlah et al. (2007) Curr. Opin.Drug Discov. Devel. 10(6):756-767; herein incorporated by reference.Dendrimers are commercially available from a number of companies,including Sigma-Aldrich (St. Louis, Mo.), Dendritech (Midland, Mich.),Starpharma (Melbourne, Australia), Polymer Factory (Stockholm, Sweden),and TCI Chemicals (Portland, Oreg.).

Negatively charged polymers that can be linked to the scaffold include,but are not limited to, negatively charged nucleic acids (e.g., DNA orRNA), peptides, polysaccharides, and poly(acrylic acid) polymers. Thepolymers linked to the scaffold, for example, may comprise one or morenegatively charged functional groups such as, but not limited to,carboxylate, sulfate, and phosphate groups. In certain embodiments, 2-10or more negatively charged polymers may be linked to the scaffold,including any number of negatively charged polymers in this range, suchas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more negatively charged polymers.

In certain embodiments, nucleic acids (e.g., DNA or RNA) are attached tothe scaffold. Generally, the length of the nucleic acids attached to thescaffold will be at least 10 nucleotides, but may range from 10nucleotides to 200 nucleotides or more including but not limited toe.g., 10 or more nucleotides, 20 or more nucleotides, 25 or morenucleotides, 30 or more nucleotides, 35 or more nucleotides, 40 or morenucleotides, 45 or more nucleotides, 50 or more nucleotides, 55 or morenucleotides, 60 or more nucleotides, 65 or more nucleotides, 70 or morenucleotides, 75 or more nucleotides, 80 or more nucleotides, 90 or morenucleotides, 95 or more nucleotides, 100 or more nucleotides, 10 to 1000nucleotides, 15 to 200 nucleotides, 20 to 200 nucleotides, 25 to 200nucleotides, 30 to 200 nucleotides, 35 to 200 nucleotides, 40 to 200nucleotides, 45 to 200 nucleotides, 50 to 200 nucleotides, 15 to 100nucleotides, 20 to 100 nucleotides, 25 to 100 nucleotides, 30 to 100nucleotides, 35 to 100 nucleotides, 40 to 100 nucleotides, 45 to 100nucleotides, 50 to 100 nucleotides, 10 to 60 nucleotides, or 15 to 50nucleotides, including any number of nucleotides in these ranges such as10, 15, 20, 25, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 57, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100,120, 140, 160, 180, 200, 400, 600, 800, or 1000 nucleotides. Exemplarymultivalent blockers having 2 to 10 nucleic acids attached to ascaffold, are described in Example 1. In certain embodiments, at leastone nucleic acid attached to the scaffold comprises a nucleotidesequence of (5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′(SEQ ID NO:1) or a nucleotide sequence displaying at least about 80-100%sequence identity thereto, including any percent identity within thisrange, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% sequence identity thereto, wherein the multivalentblocker is capable of blocking non-specific interactions with a nucleicacid of interest in a sample.

Nucleic acids (e.g., DNA or RNA) may be attached to a scaffold by anyconvenient method, as described in more detail below. The nucleic acidsmay be attached to a scaffold at any convenient point along the lengthof the nucleic acid, including at the 3′ or 5′ termini. In someinstances, a nucleic acid is attached to the scaffold at its 3′ end or5′ end. In some instances, all nucleic acid molecules are attached attheir 3′ ends to the scaffold. In some instances, all nucleic acids areattached at their 5′ ends to the scaffold.

In other embodiments, negatively charged polysaccharides are linked to ascaffold. Negatively charged polysaccharides may include those withcarboxylate groups (e.g. pectin, alginate, hyaluronan, orcarboxymethylcellulose) or sulfate groups (e.g. carrageenan, heparin, ordextran sulfate). In addition, neutral polysaccharides can be chemicallymodified with anionic groups to make them negatively charged (e.g.,carboxymethylation of pullulan or dextran, sulfation of pullulan orglucuronan).

In yet other embodiments, negatively charged peptides are linked to ascaffold. Peptides may have an overall negative charge due to thepresence of carboxylate groups from aspartate or glutamate residues. Inaddition, peptides may be modified chemically or enzymatically to addnegatively charged groups to amino acids (e.g., serine, threonine, ortyrosine phosphorylation by a kinase or tyrosine sulfation by asulfotransferase).

Negatively charged polymers can be “linked,” “conjugated,” or “attached”to or “associated” with the scaffold either covalently or noncovalently.Crosslinking agents that can be used for covalently attaching negativelycharged polymers to a scaffold include, but are not limited to, dimethylsuberimidate, N-hydroxysuccinimide, formaldehyde, and glutaraldehyde. Inaddition, carboxyl-reactive chemical groups such as diazomethane,diazoacetyl, and carbodiimide can be included for crosslinkingcarboxylic acids to primary amines. In particular, the carbodiimidecompounds, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) and N′,N′-dicyclohexyl carbodiimide (DCC) can be used forconjugation with carboxylic acids. In order to improve the efficiency ofcrosslinking reactions, N-hydroxysuccinimide (NHS) or a water-solubleanalog (e.g., Sulfo-NHS) may be used in combination with a carbodiimidecompound. The carbodiimide compound (e.g., EDC or DCC) couples NHS tocarboxyl groups to form an NHS ester intermediate, which readily reactswith primary amines at physiological pH. In addition, ultraviolet lightcan be used for linking negatively charged polymers to a scaffold. For adescription of various crosslinking agents and techniques, see, e.g.,Wong and Jameson Chemistry of Protein and Nucleic Acid Cross-Linking andConjugation (CRC Press, 2nd edition, 2011), Hermanson BioconjugateTechniques (Academic Press, 3rd edition, 2013), herein incorporated byreference in their entireties.

In certain embodiments, linking of negatively charged polymers to ascaffold is performed using click chemistry. Click chemistry can beperformed with suitable crosslinking agents comprising reactive azide oralkyne functional groups. See, e.g., Kolb et al., 2004, Angew Chem. Int.Ed. 40:3004-31; Evans, 2007, Aust. J. Chem. 60:384-95; Millward et al.(2013) Integr. Biol. (Camb) 5(1):87-95), Lallana et al. (2012) Pharm.Res. 29(1):1-34, Gregoritza et al. (2015) Eur. J. Pharm. Biopharm. 97(PtB):438-453, Musumeci et al. (2015) Curr. Med. Chem. 22(17):2022-2050,McKay et al. (2014) Chem. Biol. 21(9):1075-1101, Ulrich et al. (2014)Chemistry 20(1):34-41, Pasini (2013) Molecules 18(8):9512-9530, andWangler et al. (2010) Curr. Med. Chem. 17(11):1092-1116; hereinincorporated by reference in their entireties.

In particular, crosslinking can be performed using strain-promotedazide-alkyne cycloaddition (SPAAC) click chemistry, a Cu-free variationof click chemistry. SPAAC utilizes a substituted cyclooctyne having aninternal alkyne in a strained ring system. Ring strain together withelectron-withdrawing substituents in the cyclooctyne promote a [3+2]dipolar cycloaddition with an azide functional group. SPAAC can be usedfor bioconjugation and crosslinking by attaching azide and cyclooctynemoieties to molecules. For a description of SPAAC, see, e.g., Baskin etal. (2007) Proc. Natl. Acad. Sci. USA 104(43):16793-16797, Agard et al.(2006) ACS Chem. Biol. 1: 644-648, Codelli et al. (2008) J. Am. Chem.Soc. 130:11486-11493, Gordon et al. (2012) J. Am. Chem. Soc.134:9199-9208, Jiang et al. (2015) Soft Matter 11(30):6029-6036, Jang etal. (2012) Bioconjug Chem. 23(11):2256-2261, Ornelas et al. (2010) J.Am. Chem. Soc. 132(11):3923-3931; herein incorporated by reference intheir entireties.

In other embodiments, the negatively charged polymer and scaffold arenoncovalently linked together. For example, scaffolds with cationicgroups can bind negatively charged polymers noncovalently throughelectrostatic interactions. Alternatively, the negatively chargedpolymer and/or scaffold can be conjugated to a specific-binding moleculeor member of a binding pair to allow association of the polymer andscaffold through noncovalent binding interactions in a complex. A“binding pair” refers to first and second molecules that specificallybind to each other, such as a ligand, hormone, antagonist, or agonistand a receptor, an antigen, epitope, hapten and an antibody, or biotinand a biotin-binding protein such as streptavidin or avidin. Forexample, a negatively charged polymer can be biotinylated to allownoncovalent association of the polymer with a scaffold comprising abiotin-binding protein such as streptavidin or avidin. In anotherexample, the negatively charged polymer is modified to add an epitope(e.g., conjugated to an antigen or hapten) to allow noncovalentassociation of the polymer with a scaffold comprising an antibody. Inyet another example, the negatively charged polymer is conjugated to aligand to allow noncovalent association of the polymer with a scaffoldcomprising a receptor.

B. Applications

The multivalent blockers described herein are useful for blockingnonspecific interactions with nucleic acids and may find use in variousapplications that utilize nucleic acid materials. Multivalent blockersmay be used for blocking nonspecific interactions with any type ofnucleic acid, including DNA or RNA. For example, a multivalent blockercan be used to block nonspecific interactions with any type of RNAmolecule such as, but not limited to, messenger RNA (mRNA), transfer RNA(tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), microRNA(miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), and longnoncoding RNA (lncRNA). In another example, the nucleic acid of interestis a DNA aptamer or RNA aptamer.

Multivalent blockers can be used to block nonspecific interactions inassays that require nucleic acid binding to a target such as assaysusing aptamers (e.g. Somalogic assays), or assays requiring nucleic acidhybridization such as assays using PCR, DNA/RNA microarrays, fluorescentin situ hybridization (FISH), NanoString assays, or next generationsequencing. Multivalent blockers can also be used in assays that requireproximity of two nucleic acid probes, such as FRET, T2MR, andantibody-detection by agglutination PCR (ADAP) assays. In addition,multivalent blockers can be used in any assay that uses nucleic acidlabeled agents such as nucleic acid-antibody conjugates or nucleicacid-antigen conjugates (e.g., immuno-PCR) or other nucleic acid labeledcargos. Moreover, multivalent blockers can be used in any assaysrequiring the introduction of nucleic acid reagents to a specific sitesuch as transfection, siRNA, microRNA, CRISPR-CAS9 genome editing, andaptamer blocking.

C. Kits

The multivalent blockers described herein may be included in kits withsuitable instructions for blocking non-specific interactions withnucleic acids of interest. In addition, kits may further include samplepreparation reagents, transfection agents, detection reagents (e.g.,probes), reagents useful in amplification (e.g., PCR reagents and/orisothermal amplification reagents and/or qPCR reagents, etc.),sequencing, or performing assays (fluorescent in situ hybridization(FISH), a NanoString assay, fluorescence resonance energy transfer(FRET), T2MR, ADAP) or genome editing with nucleic acids (e.g.,CRISPR-CAS9 genome editing), buffers, diluents, etc.

The kit can comprise one or more containers for compositions containedin the kit. The kit will normally contain in separate containers thedifferent agents, including multivalent blockers and other reagents.Compositions can be in liquid form or can be lyophilized. Suitablecontainers for the compositions include, for example, bottles, vials,syringes, and test tubes. Containers can be formed from a variety ofmaterials, including glass or plastic. Instructions (e.g., written,CD-ROM, DVD, Blu-ray, flash drive, digital download, etc.) for blockingnon-specific interactions with nucleic acids will also usually beincluded in the kit.

In certain embodiments, the multivalent blocker in the kit comprises2-10 or more negatively charged polymers linked to a scaffold, includingany number of negatively charged polymers in this range, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more negatively charged polymers. Thepolymers linked to the scaffold, for example, may comprise one or morenegatively charged functional groups such as, but not limited to,carboxylate, sulfate, and phosphate groups. The negatively chargedpolymers can be linked to the scaffold covalently or noncovalently.

In certain embodiments, the polymers linked to the scaffold are nucleicacids. For example, 2-10 or more nucleic acids may be linked to thescaffold, including any number of nucleic acids in this range, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acids. In certainembodiments, the kit comprises a multivalent blocker comprising at leasttwo nucleic acids linked to a scaffold, wherein at least one nucleicacid comprises the nucleotide sequence of(5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ ID NO:1),or a sequence displaying at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%sequence identity thereto, wherein the multivalent blocker is capable ofblocking non-specific interactions with a nucleic acid of interest in asample.

In other embodiments the polymers linked to the scaffold are negativelycharged polymers, such as poly(acrylic acid) polymers. For example, 2-10or more negatively charged polymers may be linked to the scaffold,including any number of negatively charged polymers in this range, suchas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more negatively charged polymers.

In certain embodiments, the kit comprises a multivalent blockercomprising a scaffold comprising a dendrimer (e.g., polyamidoamine(PAMAM) dendrimer), a multi-armed polyethylene glycol (PEG), ananoparticle (e.g., gold nanoparticle), or a protein (e.g., streptavidinor avidin).

In another embodiment, the kit further comprises one or more reagentsfor performing real time quantitative PCR, microarray analysis,fluorescent in situ hybridization (FISH), a NanoString assay, nextgeneration sequencing, fluorescence resonance energy transfer (FRET), T2magnetic resonance (T2MR), antibody-detection by agglutination PCR(ADAP), or CRISPR-CAS9 genome editing, or transfection.

3. Experimental

This section provides greater detail on desirable embodiments forcarrying out the present invention.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Blocking Non-Specific Interactions with Nucleic Acids

Description of invention: Several common biological compounds are knownto facilitate unwanted interactions with nucleic acids. These substancesinclude fibrinogen, fibrin degradation product, cell debris includingnucleoproteins and histones [11]. Many of these molecules are positivecharged, permitted electrostatic interaction with negatively chargednucleic acids. Reported binding constants between these substances andnucleic acids vary widely. In addition, these substances can exist inbiological samples in wide concentration ranges (pM to mM). Lab plasticsalso adsorb nucleic acid molecules, facilitating non-specificinteractions and sequestration. A universal blocking agent ideallyshould neutralize all these types of interfering compounds.

Currently-used blocking materials fail to neutralize all potential sitesof non-specific binding. Thus, functional and useful nucleic acids arestill vulnerable to nonspecifically bind with interfering substances. Wehypothesized that the failure of state of the art blocking agents iscaused by their limited affinity for the non-specifically bindingsubstances. A state of the art blocker may engage with non-specificbinding sites temporarily, then dissociate, and re-associate. However,during the period when blocking agents dissociate, precious nucleic acidmaterials may be bound at non-specific binding sites.

Here we describe a substantially improved method to block and preventnonspecific interactions between nucleic acids and other molecules, andprovide experimental evidence showing that our method is more effectivethan other current methods. We invented a highly potent nucleic acidblocking agent that engages with non-specific binding sites withsubstantially enhanced avidity, thereby reducing the possibility ofdissociation from the non-specific binding sites (FIGS. 1 and 2).

Our blocking agent is a multi-valent nucleic acid material, which isproduced by linking two or more nucleic acids onto a common scaffold. Wedemonstrated the scaffold could be organic molecules (such as adendrimer), inorganic molecules (nanoparticles), or biological molecules(such as streptavidin). We also demonstrated the linkage between thenucleic acid and the scaffold could be either covalent or non-covalent.We demonstrate the multi-valency alone is the major critical factor inenhancing blocking efficacy. Taken together, this invention employsmulti-valent nucleic acids as a new generation of blocking agents topermanently and completely engage charged surfaces of interferingspecies, thereby eliminating non-specific interactions.

Alternatively, negatively charged polymers can be used in synthesis of amultivalent blocker that has two or more arms of negative charges. Thistype of multi-valent blocker should also be able to engage withnon-specific nucleic acid binding sites with high avidity (FIG. 2).

Indeed, multi-valency is a common molecular strategy to increase bindingaffinity between proteins. For instance, a monovalent majorhistocompatibility complex (MHC)-peptide complex only weakly interactswith the T-cell receptor, making it difficult to for labeling orimaging. By assembling biotinylated MHC-peptide complexes into atetramer through the use of streptavidin, the interaction of thetetrameric ensemble with T-cell receptors is greatly enhanced,permitting labeling for further experiments [10].

Our multi-valent nucleic acid blocker is inspired by the aforementionedexample. We hypothesized and validated with evidence described belowthat multi-valent nucleic acid materials could be used to boost bindingavidity with non-specific nucleic acid binding sites, thus achievingmuch improved reduction of non-specific binding in comparison to currentstate of the art methods.

Evidence:

To assure our multi-valent nucleic acid blocker indeed blocksnonspecific interactions and improves performance in comparison to stateof the art methods, we tested their impact in a nucleic acid-basedimmunoassay, termed antibody-detection by agglutination-PCR (ADAP) assay[5]. In this assay, antigen-DNA conjugates probes are used to detectpresence of target antibodies in a biological specimen. Here, we usedgreen fluorescent protein (GFP)-DNA conjugates as negative controlprobes. As human samples should not contain antibodies against GFP, weexpect that all human samples should generate signal very close to thebaseline (or hopefully no signals at all). However, when assaying largebatches of human samples, we consistently identified samples thatgenerated strong signals in the presumed absence of anti-GFP antibodies.These results indicated the presence of non-specific nucleic acidinteractions.

To identify blocking agent that could reduce the background signals, wefirst used blocking agents such as synthetic single-stranded DNA (60 bp)at very high quantity (10 mg/mL). However, no improvement of backgroundsignals was observed. Similarly, the addition of extracted salmon spermDNA at high concentration (5 mg/mL) does not reduce the background. Incontrast, with the addition of a multi-valent single-stranded DNAblocker, we saw complete elimination of background signals, suggestingthat this material has drastically improved blocking properties comparedto state of the art methods (FIG. 6).

To further investigate whether this effect is indeed a result ofmulti-valency, we performed series of experiments. First, we synthesizedthree different conjugates with different valencies: 2, 5 or 10 DNAstrands per dendrimer molecule (FIG. 5). In each experiment, we loadedthe multi-valent blocker at the same DNA concentration into the system,so that differences in blocking efficiency could not trivially beascribed to increased DNA content (FIG. 9). Conjugates with DNA ratiosof 5:1 and 10:1 showed improved blocking activity compared to the 2:1DNA ratio conjugate. The similar blocking efficiency of 5:1 and 10:1 DNAratio conjugates implies that further increasing of multi-valency mightnot have measurable outcomes. Notably, 2:1 DNA ratio conjugates exhibitsubstantially higher blocking activity than normal ssDNA alone. Theseobservations suggest that multi-valency is driving the blockingefficacy.

However, one might argue that this phenomenon is unique todendrimer-based nucleic acid conjugates and not a result ofmulti-valency. For instance, one could argue that the unique linkage inthe conjugation of nucleic acid and dendrimer is contributing to theblocking effect. Or one could argue the presence of a dendrimer rendersthe nucleic acid into a unique conformation, thereby enhancing theblocking efficacy. To preclude these possibilities, we synthesized aseries of different multi-valent nucleic acids materials (FIG. 8). Theseincluded nucleic acids conjugated to a small dendrimer core (G.3, 7 kD,2 nm size), a large dendrimer core (G6, 58 kD, 5 nm size), goldnanoparticles and streptavidin. Satisfyingly, all these differentmulti-valent blocking agents showed substantial improved blockingeffects compared to state of the art methods (e.g. free single-strandedDNA, salmon sperm DNA and detergents). We used core scaffold materialswith different sizes, different properties (organic molecules, inorganicmolecules, proteins) and different linkages (co-valent or non-covalent)to unambiguously demonstrate that the only common feature among theseblockers is the multi-valency of the nucleic acids. Therefore, strongevidence is provided that the multi-valency of the nucleic acid-basedblocking agents is the major driver of the blocking effect.

Interestingly, among these four types of blocking materials, we observedthat the G3 and G6 dendrimers share similar blocking effects (99%), goldnanoparticles (57%) and streptavidin (58%). Although we cannotexplicitly confirm why dendrimer-based conjugates had superior blockingeffects, we hypothesize that the weaker effects of streptavidin-basedmaterials are because they are limited to tetrameric multi-valency atmost, as streptavidin only has 4 biotin-binding pockets. The goldnanoparticles may also suffer as gold nanoparticles aggregated togetherduring the testing process, a common behavior of these nanoparticles.

In summary, a general strategy is described to prevent non-specificbinding with nucleic acid materials (FIGS. 1-4). Blocking efficiency canbe markedly increased by linking multiple (2 or more) nucleic acidstogether to form multivalent nucleic acid blockers. The nucleic acidportion in the nucleic blocker should carry sequences that do notinterfere with the intend use. Alternatively, the nucleic acid portionof the multivalent blocker can be substituted by materials that carrynegative charges such as an acrylic acid polymer. The core concept is tolink multiple negatively charged materials onto a common scaffold toform a multi-valent blocker capable of blocking non-specific chargeinteractions with nucleic acid materials.

Furthermore, to further enhance the blocking performance, furthermodifications may be made to the multivalent blocker to make the blockermore similar to the actual nucleic acid probe (FIG. 3). For instance, ifthe actual nucleic acid probe carries a fluorescent tag, we cansynthesize a multi-valent blocker that has the fluorescent tags attachedso that the multivalent blocker also prevents non-specific binding tothe fluorescent probe portion.

The multivalent blockers described in this invention can be used foressentially any biological system that uses nucleic acid materials. Forinstance, multivalent blockers can be used to block nonspecificinteractions in assays that require nucleic acid binding to a targetsuch as assays using aptamers (e.g. Somalogic assays) [2, 4], or anyassay requiring nucleic acid hybridization such as assays using DNA/RNAmicroarrays, fluorescent in situ hybridization (FISH), NanoString assays[3], or next generation sequencing [9]. Multivalent blockers can also beused in any assays that require proximity of two nucleic acid probes,such as FRET, T2MR [6], and antibody-detection by agglutination PCRassays [6]. In addition, multivalent blockers can be used in any assaythat uses nucleic acid labeled agents such as nucleic acid-antibodyconjugates [9], nucleic acid-antigen conjugates or any other nucleicacid labeled cargos. These reagents are commonly used in immune-PCR andsingle-cell studies. Moreover, multivalent blockers can be used in anyassays requiring the introduction of nucleic acid reagents to a specificsite such as transfection, siRNA, microRNA [1], CRISPR-CAS9 assays andaptamer blocking.

The following examples and experimental methods are offered forillustrative purposes only, and are not intended to limit the scope ofthe present invention in any way.

EXAMPLES Experimental Methods

(1) Preparation of multi-valent nucleic acid blocking agent. To test thefeasibility of using multi-valent nucleic acid to block non-specificinteractions, we synthesized multi-valent nucleic acid reagents.Briefly, we used single-stranded DNA oligonucleotides with the followingsequences as the blocking nucleic acids (Block seq:5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ ID NO:1)).We synthesized multi-valent blocking agents using a dendrimer scaffold(FIG. 5). Dendrimers are organic molecules with repeated branches. Herewe used a dendrimer based on polyamidoamine (PAMAM). PAMAM dendrimershave multiple primary amine terminal groups (amino-functional group(NH2-)), which can serve as linkage points with nucleic acids. Here, weused generation 3 PAMAM as the dendrimer core. The generation 3dendrimer core is a relatively small molecule with a molecular weight of6909, size of 3.6 nm and has 32 terminal amine groups on its surface. Weused sulfo-SMCC (Sulfo-SMCC (sulfosuccinimidyl 4-(N15maleimidomethyl)cyclohexane-1-carboxylate)) as a crosslinker. Weincubated the PAMAM dendrimer with excess sulfo-SMCC for 2 hours at roomtemperature (RT). The succinimidyl-part of sulfo-SMCC reacted with theterminal amine functional groups on the dendrimer. Then, we used a sizeexclusion column to remove unreacted sulfo-SMCC from the sulfo-SMCCactivated dendrimer. Then, we incubated the activated dendrimer with thethiolated nucleic acids (e.g., 5′thiol-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ IDNO:1)) overnight at 4° C. Importantly, the thiolated nucleic acidsneeded to be pre-reduced with dithiothreitol (DTT) to reduce theoxidized thiolated nucleic acids. The reduced thiol functional group ofthe nucleic acid reacted with the maleimide functional group on theactivated SMCC-dendrimer. We could modify the incubation ratio betweennucleic acids and activated dendrimer to fine tune the final DNAmulti-valency. For instance, we could incubate the nucleic acid anddendrimer at 2:1, 5:1, and 10:1 ratios to make correspondingmulti-valent nucleic acid blockers. The final DNA ratios were validatedby gel analysis. Typically, in this reaction, one would see adistribution of DNA ratios. For instance, if DNA is incubated with adendrimer at a 5:1 ratio, the major product produced has 5 DNA perdendrimer, but we also observed some minor bands on the gels for sideproducts having 3:1, 4:1, 6:1, and 7:1 DNA:dendrimer ratios. Generally,the final DNA ratio on average was close to the incubated DNA todendrimer ratio.

(2) Validate Blocking Efficiency of Multi-Valent Nucleic Acids.

To test the blocking efficiency, we used a published antibody-detectionby agglutination-PCR (ADAP) assay as a proof of principle assay (ACSCent. Sci., 2016, 2 (3), pp 139-147). Briefly, this assay usedantigen-DNA conjugates to detect presence of antibodies in a testsample. We used GFP (green fluorescent protein)-DNA conjugates in anADAP assay to screen 50 patient sera. Theoretically, human patientsshould not have antibodies against GFP antigens. Therefore, one wouldexpect no signals should be observed for these 50 patient sera. However,we identified 5 patient sera that generated very strong ADAP signals. Weattributed these strong signals to non-specific interactions betweensubstances in patient sera and the GFP-DNA conjugate probes. Then, wecompared three different common blocking agents and the multi-valentnucleic acid blocking agent for their capabilities to reduce thenon-specific interactions. Briefly, we used single-stranded DNA thatcontained the same block seq(5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ ID NO:1))at final concentration of 100 μM as the blocking agent. Or we useddouble-stranded DNA of the same sequences at final concentration of 100μM as blocking agent. In addition, we used salmon-sperm DNA at finalconcentration of 1 mg/mL or dextran sulfate at a final concentration of0.25 mg/mL as blocking agents. The concentrations we used were either onthe same or slightly higher than common literature values. Thus, ifthese substances were effective, we should have seen the effects veryevidently. Finally, we also used a multi-valent nucleic acid blockermade from a generation 3 dendrimer at a final concentration of 16 μM(the concentration is referring to the total concentration ofsingle-stranded DNA). In other words, we incubated 1 μL of patient serumand 2 μL of probe mix (contains either none or one of the above blockingagents, 400 pM GFP-DNA conjugates, 4% BSA, 100 mM NaCl, lx PBS pH=7.4)for 30 minutes at 37° C. Then, we performed the ligation step,pre-amplification step and final real-time quantitative PCR (qPCR) stepfollowing published ADAP protocols. The tests results are shown in FIG.6. We observed that single-stranded DNA, double-stranded DNA andsalmon-sperm DNA provided no improvement in reducing non-specificsignals. Dextran sulfate reduced non-specific signals by 50%, whereasthe multi-valent nucleic acid blockers reduced non-specific signalscompletely. Therefore, this data demonstrates that multi-valent blockingagents are superior in blocking non-specific interactions with DNA-basedprobes.

Critically, it is important to ensure blocking agents did not interferewith the assay performance. Therefore, we performed a separate ADAPexperiment using p24-DNA conjugates and tested a dilution series ofstrongly positive HIV patient serum (which contains anti-p24antibodies). We compared the signal intensities across dilution seriesbetween assays performed with the dextran sulfate and the multi-valentnucleic acid blocker (FIG. 7). We observed that the dextran sulfateyielded significantly reduced signals (thus lower assay sensitivity)compared to the multi-valent nucleic acid blocker. The reduced signalswith dextran sulfate could be attributed to its inhibitory effect on PCRreactions, which has been widely reported in the literature. In summary,we used these experiments to demonstrate that multi-valent blockers aresuperior than current blocking agents in blocking non-specificinteractions in nucleic acid-based assays without interfering with assayperformance.

(3) Synthesizing Alternative Multi-Valent Blocking Agents.

In order to validate that the observed superior blocking capabilitiesare not unique to dendrimer-based nucleic acid conjugates, we preparedseveral different multi-valent blocking agent constructs. Weinvestigated different core materials to see if we could still seesimilar efficient blocking behavior. We sought to confirm that theobserved blocking capability is a result of multi-valency. All thesingle-stranded DNA we used in this experiment shared the same blockingsequences as in experiment 1.

(5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ ID NO: 1))

First, we purchased another dendrimer of larger size. We obtainedgeneration 6 PAMAM dendrimer. This dendrimer has a molecular weight of58 kD, size of 6.7 nm and 256 amino terminal groups. We then synthesizedsingle-stranded DNA dendrimers following protocols outlined inexperiment 1.

Secondly, we synthesized gold nanoparticles DNA conjugates. It is widelyreported in the literature that thiolated DNA can be readily conjugatedto gold nanoparticles. Therefore, we purchased gold nanoparticles of 2nm and 20 nm in size. Then, we incubated these nanoparticles withthiolated single-stranded DNA. Again, the thiolated DNA had been reducedby DTT, and excess DTT was removed using a spin column (Thermo Fischer,Zeba spin desalting column, 7 kD cutoff). We incubated the DNA andnanoparticles at a 200:1 ratio with constant rotating at roomtemperature overnight. Then, we used centrifugation to pellet the goldnanoparticles and removed excess DNA to obtain pure DNA-nanoparticleconjugates. The successful conjugation was validated by UV-VISspectroscopy and gel analysis. Then, we concentrated the DNA-goldnanoparticle solution to a final DNA concentration of 50 μM.

Thirdly, we synthesized streptavidin nucleic acid conjugates byincubating streptavidin with biotin-labeled single-stranded DNA. Thestreptavidin binds biotin-label substances with strong affinity (Kd<1nM). Therefore, the streptavidin DNA conjugates could be synthesizedreadily by incubation with the biotin-labeled single-stranded DNA.

(4) Comparing Performance of Different Multi-Valent Nucleic AcidBlockers.

Then, we tested the blocking capacity of these different multi-valentnucleic acid blockers following protocols outlined in experiment 2.Briefly, we incubated 1 μL patient serum (that showed strong signalsusing negative control GFP-DNA conjugates) with 2 μL probe mix (thatcontain 400 pM GFP-DNA conjugates, one of the above multi-valent blockerat 16 μM, 100 mM NaCl, lx PBS, pH=7.4). To fairly compare performance ofdifferent blockers, the blocker concentration is referring to thesingle-stranded DNA concentration. Thus, all blocking agents were loadedat concentrations so as to contain the same amount of single-strandedDNA. Then, we processed the samples by performing subsequent ligation,pre-amplification, and qPCR quantification steps. The results are shownin FIG. 8. It is evident that all multi-valent blocking agents were moreefficient than single-stranded DNA alone and could reduce non-specificsignals by more than 50%. This result demonstrates that variousdifferent multi-valent blocker constructs could be used to reducenon-specific binding, and strongly supports the notion that multivalencyof nucleic acid is the major driver for improved blocking ability.

Furthermore, we also compared the performance of multivalent blockerswith different valencies (FIG. 9). We observed that bivalency (2 DNA perblocker) alone had markedly enhanced blocking capabilities thansingle-stranded DNA alone. The increase in blocking efficiency seemed toplateau at pentavalency (5 DNA per blocker). This result furthersupports the notion that multivalency is indeed driving blockingefficiency.

REFERENCES

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Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined herein.

1. (canceled)
 2. A method of blocking non-specific interactions with anucleic acid of interest in a sample, the method comprising contactingthe sample with a multivalent blocker, said multivalent blockercomprising: (a) at least two nucleic acids linked to a non-proteinscaffold; or (b) at least two non-nucleic acid negatively chargedpolymers linked to a scaffold, wherein the multivalent blocker binds topositively charged compounds or materials in the sample, therebyblocking non-specific interactions with the nucleic acid of interest. 3.The method of claim 2, wherein the at least two non-nucleic acidnegatively charged polymers comprise carboxylate, sulfate, or phosphate,and are linked to the non-protein scaffold.
 4. The method of claim 2,wherein said scaffold comprises a dendrimer, multi-armed polyethyleneglycol (PEG), or a metal nanoparticle.
 5. The method of claim 2, whereinsaid scaffold comprises a dendrimer and said dendrimer is a polylysinedendrimer, a poly(amidoamine) (PAMAM) dendrimer, a poly(propylene imine)dendrimer, or a poly(etherhydroxylamine) (PEHAM) dendrimer.
 6. Themethod of claim 2, wherein the nucleic acids are linked to the scaffoldcovalently.
 7. The method of claim 2, wherein the nucleic acid ofinterest is a nucleic acid probe comprising a detectable label.
 8. Themethod of claim 7, wherein the multivalent blocker further comprises thesame detectable label as the nucleic acid probe.
 9. The method of claim2, further comprising performing real time quantitative polymerase chainreaction (RT-PCR), microarray analysis, fluorescent in situhybridization (FISH), a NanoString assay, next generation sequencing,fluorescence resonance energy transfer (FRET), T2 magnetic resonance(T2MR), antibody-detection by agglutination PCR (ADAP), CRISPR-CAS9genome editing, or transfection while blocking the non-specificinteractions with the nucleic acid of interest with the multivalentblocker.
 10. The method of claim 2, wherein the nucleic acid of interestis conjugated to an antibody, a lipid, a carbohydrate, a nanoparticle,or a cationic molecule.
 11. The method of claim 2, wherein the nucleicacid of interest is DNA or RNA.
 12. The method of claim 11, wherein theRNA is selected from the group consisting of messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA(siRNA), microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA(snRNA), and long noncoding RNA (lncRNA).
 13. The method of claim 2,wherein the nucleic acid of interest is a DNA aptamer or RNA aptamer.14. A composition comprising a multivalent blocker, which comprises: (a)at least two nucleic acids linked to a non-protein scaffold; or (b) atleast two non-nucleic acid negatively charged polymers linked to ascaffold, wherein the multivalent blocker binds to positively chargedcompounds or materials in a sample.
 15. The composition of claim 14,wherein the at least two non-nucleic acid negatively charged polymerscomprise carboxylate, sulfate, or phosphate, and are linked to thenon-protein scaffold.
 16. The composition of claim 14, wherein saidscaffold comprises a dendrimer, multi-armed polyethylene glycol (PEG),or a metal nanoparticle.
 17. The composition of claim 14, wherein saidscaffold comprises a dendrimer and said dendrimer is a polylysinedendrimer, a poly(amidoamine) (PAMAM) dendrimer, a poly(propylene imine)dendrimer, or a poly(etherhydroxylamine) (PEHAM) dendrimer.
 18. Thecomposition of claim 14, wherein at least one nucleic acid is selectedfrom: (a) a nucleic acid comprising a nucleotide sequence of(5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ ID NO:1);or (b) a nucleic acid comprising a nucleotide sequence having at least81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99% identity to the sequence of(5′-TCGTGGAACTATCTAGCGGTGTACGTGAGTGGGCATGTAGCAAGAGGGTC-3′ (SEQ ID NO:1).19. A kit comprising the composition of claim 14 and instructions forusing a multivalent blocker for blocking non-specific interactions withnucleic acids in a sample.
 20. The kit of claim 18 further comprisingreagents for performing real time quantitative polymerase chain reaction(RT-PCR), microarray analysis, fluorescent in situ hybridization (FISH),a NanoString assay, next generation sequencing, fluorescence resonanceenergy transfer (FRET), T2 magnetic resonance (T2MR), antibody-detectionby agglutination PCR (ADAP), or CRISPR-CAS9 genome editing, ortransfection.